Early immune profiling reveals distinct inflammatory responses between children and adults few days after primary SARS-CoV-2 infection

Abstract

Background: To date, it is still not clear why during the COVID-19 pandemic children generally developed no or milder symptoms compared to adults. As innate immune responses are crucial in the early defense against pathogens, we aimed at profiling these responses from both adults and children with a primary SARS-CoV-2 infection.

Methods: In the first months of the pandemic, PBMCs and serum were collected from peripheral blood of adults and children at different time points after testing SARS-CoV-2 PCR positive (PCR+). The levels of SARS-CoV-2 Spike-specific IgG were measured in serum. The cells were cultured for 24 hours in medium only, with heat inactivated SARS-CoV-2 (iSARS-CoV-2) or toll-like receptor (TLR) ligands. The levels of secreted cytokines/chemokines as well as monocyte phenotype were determined.

Results: Few days after testing PCR+, PBMCs from PCR+ children secreted higher levels of cytokines/chemokines compared to PCR+ adults, after these cells were incubated either in medium only or after stimulation with iSARS-CoV-2 or TLR ligands. Furthermore, PBMCs from children stimulated with iSARS-CoV-2 secreted significantly higher levels of IL-10 and GM-CSF compared to PBMCs from control children. In contrast, PBMCs from the PCR+ adults secreted lower levels of IL-8 compared to adult controls. Phenotypic analysis of monocytes indicates a smaller proportion non-classical monocytes for adults compared to children. The distinct cytokine profiles, symptom severity, and the proportion of non-classical monocytes correlated to each other. The levels of Spike-specific IgG overtime did not significantly differ between children and adults.

Conclusions: Within the first week after testing PCR+, children showed a stronger inflammatory innate immune profile and experienced less severe symptoms compared to adults. Our data implies correlations between the secretion of cytokines/chemokines, proportion of non-classical monocytes, and symptoms severity. These findings enhance our understanding of the distinct pediatric and adult innate immune profile after SARS-CoV-2 infection and contributes to the knowledge necessary to improve future prevention strategies.

Keywords: COVID-19; age; innate immune response; monocytes; toll like receptors.

Figure 1

Divergent cytokine and chemokine profile induced by iSARS-CoV-2 in PBMCs from healthy control and recently infected children and adults. Cytokine and chemokine levels were measured in culture supernatant from PBMCs after overnight stimulation with iSARS-CoV-2 (MOI=3). Kruskal-Wallis non-parametric test was used to compare healthy control (HC) to PCR+ individuals among children (▲) or adults (○), as well as HC or PCR+ children to their counterparts in adults, after which Dunn’s multiple comparison test was executed. Error bars indicate median value with 95% CI. *p-adj<0.05. **p-adj-<0.01.

Figure 2

The development of anti-SARS-CoV-2 Spike-specific antibodies over time in PCR+ children and adults. SARS-CoV-2 Spike-specific IgG levels were measured within <7 days (T1), 2-3 weeks (T2), 4-6 weeks (T3) after testing PCR+ in serum of children (A) and adults (B). IgG levels are expressed as binding antibody units (BAU)/ml. Dotted line indicates seropositivity threshold of 10.1 BAU/ml.

Figure 3

Symptom severity correlates with cytokine/chemokine production. Cytokine and chemokine levels delineated by symptom severity groups in adults (A) and children (B). Kruskal-Wallis non-parametric with Dunn’s multiple comparison test was performed comparing each group to each other. Error bars indicate median value with 95% CI. *p-adj<0.05. **p-adj<0.01.

Figure 4

Monocyte subset proportions differ between PCR+ children and adults. Flow cytometric analysis of monocytes within PBMCS after iSARS-CoV-2 stimulation. Proportions of classical monocytes (CD14+CD16-), intermediate monocytes (CD14+CD16+), and non-classical monocytes (CD14lowCD16+) among total monocytes in PBMCs. Kruskal-Wallis non-parametric test was used to compare HC to PCR+ individuals among children (▲) or adults (○), as well as HC or PCR+ children to their counterparts in adults, after which Dunn’s multiple comparison test was executed. Error bars indicate median value with 95% CI. **p-adj-<0.01. ***p-adj<0.001.

Figure 5

PBMCs from PCR+ children secret more cytokines and chemokines after TLR activation compared to adults. Cytokines and chemokines were measured in supernatant of PBMCs from PCR+ children (▲) and adults (○) after overnight stimulation of TLR2 with HKLM, TLR4 with lipopolysaccharide (LPS), or TLR7/8 with Resiquimod (R848). Values are shown as log10 pg/ml. Multiple Mann-Whitney test with Bonferroni-Dunn multiple comparison test were performed to compare response by children and adults for each stimulation or mock. Error bars indicate median value with 95% CI. *p-adj<0.05. **p-adj<0.01. ***p-adj<0.001.

Figure 6

Adult PBMCs produce more cytokines and chemokines at later time points post infection. Cytokines and chemokines were measured in culture supernatant after iSARS-CoV-2 (A), HKLM (B), LPS (C), or R848 (D) stimulation of adult PBMCs collected at T1 (<7days), T2 (2-3 weeks), and T3 (4-6 weeks) post infection. Dotted line indicates average value of healthy control group. Friedmans test for non-parametric paired samples. followed by Dunn’s multiple comparison test was used to compare responses at each time point. *p-adj<0.05. **p-adj<0.01. ***p-adj<0.001.